1. Field of the Invention
The present invention relates to assays, both immunoassays and nucleic acid assays of analytes, which utilize a universal detection system based on polynucleotide interactions.
2. Description of the Prior Art
The analysis and detection of minute quantities of substances in biological and non-biological samples has become a routine practice in clinical and analytical laboratories around the world. Broadly, the analytical techniques can be divided into those based on ligand-receptor interactions (e.g., immunoassay-based techniques), and those based on nucleic acid hybridization (polynucleotide sequence-based techniques).
For example, immunoassay techniques involve, at some stage or step in the process, the non-covalent association between an antibody binding site and an antigen complementary therefor. See, for example, “An Introduction to Radioimmunoassay and Related Techniques” by T. Chard, Elsevier Science Publishers B.V. (Biomedical Division), Amsterdam, N.Y., Oxford, 1978. In polynucleotide sequence-based techniques, the process, at some step or another, involves the non-covalent binding of a polynucleotide sequence to a complementary sequence under hybridization conditions. (See for example, Falkow et al, U.S. Pat. No. 4,358,535, Wahl et al, U.S. Pat. No. 4,302,204, and Heimer, U.S. Pat. No. 3,755,086.)
In a generalized sense, both the aforementioned techniques involve a primary recognition event, brought about by precise molecular alignment and interaction, and energetically favored by the release of non-covalent bonding free energy (e.g., hydrogen bonding, dispersion bonding, ionic bonding, dipolar bonding, and the like). In addition to the primary recognition event, both techniques also involve at one step or another, a signalling event. This step or event relates to the necessity of detecting, in some demonstrable manner to a human or instrument detection system, the primary recognition event.
Signalling has been centered mainly in two broad areas: radioactive and non-radioactive techniques. Radioactive signalling has relied on radiolabeling of one or more components involved in the system, with such atoms as 32P, 131I, 14C, 3H, and the like. Detection is usually by means of a radioactivity detector. Non-radioactive techniques have been increasingly used in the last few years, since they involve no radioactivity, thus making such techniques safer, cleaner and more stable towards storage. They have been developed to sensitivities as high if not higher than radiolabeling techniques. Among the most common non-radioactive signalling techniques used at present are enzyme linked immunoassays (see, for example, Schuurs, A. H. et al, Clinica Chimica Acta, 81: 1-40 (1977)), fluorescence (Bauman et al, Chromosoma, 84: 1-18 (1981)), indirect immunofluorescence (Rudkin et al, Nature, 265: 472-473 (1977)), avidin-biotin interactions (Manning, J, et al, Biochemistry, 16: 1365-1370 (1977)), electron microscopy of electron dense nuclei such as ferritin (Broker, T. R. et al, Nucleic Acids Research 5: 363-384 (1978)), latex attachment (Sodja, A., ibid 5: 385-401 (1978)), combinations of the aforementioned techniques, and others.
The primary recognition event and the signalling event need to be coupled to each other, directly or indirectly, proportionately or inversely proportionately. Thus, in such systems as nucleic acid hybridizations with radiolabeled probes, the amount of radioactivity is usually directly proportional to the amount of analyte present. The same is true with a system such as a sandwich immunoassay, wherein the amount of labeled second antibody being detected is normally directly proportional to the amount of antigen present in the sample. Inversely proportional techniques include, for example, competitive immunoassays, wherein the amount of detected signal decreases the more analyte present in the sample.
The prior art has also utilized amplification techniques, wherein the signalling event is related to the primary recognition event in a ratio greater than 1:1. Thus, the signalling component of the assay may be present in a ratio of 10:1 to each recognition component, thereby providing a 10-fold increase in sensitivity.
The great versatility polynucleotide sequence-based recognition systems has caused an extensive amount of experimentation and research to be invested therein. This versatility is brought about by the precise alignment of complementary nucleotide bases to each other, adenine (A) aligning to thymidine (T) and guanine aligning with cytidine (C). Given this complementarity, it is possible to utilize any desired sequence to provide an infinitely versatile system.
One of the impediments to a more extensive use of polynucleotide interaction-based systems, however, has been the necessity of attaching signalling or reporter groups (such as radioactive phosphorous, or enzymes, or biotin, or the like) to individual nucleotide residues in the polymer chain. At least two problems arise out of this requirement.
First, the chemical reaction conditions involved in the modification of a polynucleotide polymer are generally too vigorous to be sufficiently selective for any one nucleotide in particular. For example, dicarbonyl reagents such as kethoxal or glyoxal will indiscriminately react with guanine (see for example Shapiro, R. et al, Biochemistry, 5: 2799-2807 (1966), Litt, M, ibid, 8: 3249-3253 (1969), or Politz, S. M. et al, ibid, 20: 372-378 (1981)). Thus, if one were to use a dicarbonyl-based cross-linking agent to attach an enzyme or a low molecular signalling compound directly on a polynucleotide chain, one would risk (and in fact one obtains) modification of a substantial amount of all guanine residues in the chain. This, of course, severely hinders the use of such modified chain in a recognition step. This problem has been solved in the prior art by the use of enzymatic (DNA polymerase-based) incorporation of individual modified nucleotides (previously modified in a non-hydrogen-bond-disruptive manner) into a nascent polynucleotide chain. It would, however, be preferred to utilize chemical modification techniques on the final polynucleotide polymer itself.
A second problem is associated with the attachment of signalling groups to polynucleotides and is somewhat related to the first. The problem is based on the necessity of synthesizing, by sometimes sophisticated and elaborate synthetic techniques, the modified monomeric nucleotide units themselves, prior to their enzymatic incorporation into polymer. Thus, radiolabeled nucleotides or biotin-labelled nucleotides have to be independently synthesized. Further, the amount of incorporation of a chemically modified nucleotide into final nucleic acid polymer may also influence the ability of a probe to recognize a given sequence on the analyte. This is particularly important if amplification techniques are utilized wherein signalling groups greatly outnumber recognition groups.
It would therefore be very useful to develop an assay system which utilizes components that are easily prepared, amenable to chemical modification rather than enzymatic-based reactions, that would utilize the great versatility of polynucleotide-based sequence recognition, and include the possibility of signal amplification methods.